The cloning and expression of foreign genes in bacteria and yeast has been demonstrated to be a viable means for the production of a variety of useful proteins. Expression of foreign genes in these microorganisms has relied on the use of autonomously replicating extrachromosomal elements, generally known as plasmids. However, these expression systems have presented numerous problems for industrial scale-up, including problems in protein solubility, purification, and secretion.
Filamentous fungal genera, such as Aspergillus and Penicillium, have a long history of industrial use in the manufacture of enzymes and specialty chemicals. The development of these organisms as industrially important producers has heretofore relied on classical methods of mutation followed by screening to obtain suitable high producing strains.
In light of this fermentation technology, it would be beneficial to develop an efficient system for expressing foreign genes in filamentous fungi. The industrial use of filamentous fungi could then be expanded to include pharmaceuticals, enzymes, and other products that are not made, or are made only inefficiently by these fungi.
Filamentous fungi present several other potential advantages over bacteria and yeast. For instance, species of filamentous fungi are known which secrete a wide variety of substances, a feature which facilitates the purification of those products. Furthermore, these organisms do not exhibit the extreme codon usage bias of yeast, which may make them more suitable for the production of some foreign proteins. Additionally, research has suggested that some species are also capable of processing intervening sequences present within the genes of higher eucaryotes, in a manner similar to higher eucaryotes, which bacteria and yeast cannot do. Filamentous fungi are also known to effect post-translational modifications of proteins, such as glycosylation and phosphorylation, in a manner similar to higher eucaryotes, which may be important to the efficacy of the proteins.
Transformation systems for filamentous fungi have been described by a number of authors. In the case of the genus Aspergillus, these systems rely on chromosomal integration of the cloned DNA. Although autonomously replicating extrachromosomal elements have been utilized to obtain expression of a heterologous gene in Neurospora (Lambowitz, U.S. Pat. No. 4,486,533), Aspergillus species generally lack extrachromosomal plasmid elements and it is believed that such elements would not function adequately in these fungi.
Reports of transformation in filamentous fungi have generally been directed to complementing mutations with wild-type DNA from the same species. Yelton et al. (Proc. Natl. Acad. Sci. USA 81: 1470-1474, 1984) transformed a trpC.sup.- strain of Aspergillus nidulans to trpC.sup.+ using a plasmid containing the complete A. nidulans trpC gene, and similarly, an argB.sup.- strain was transformed to argB.sup.+. Tilburn et al. (Gene 26: 205-221, 1983) transformed A. nidulans using the cloned amdS gene. Yelton et al. (Proc. Natl. Acad. Sci. USA 82: 834-838, 1985) used an A. nidulans cosmid library to complement several mutations in an A. nidulans strain.
Several authors have also described transformation of filamentous fungal species with DNA from closely related fungal species. Ballance et al. (Biochem. Biophys. Res. Comm. 112: 284-289, 1983) transformed an A. nidulans pyrimidine auxotroph to prototrophy using the orotidine-5'-phosphate decarboxylase gene of Neurospora crassa. Kelly and Hynes (EMBO J. 4: 475-479, 1985) transformed wild type strains of A. niger with the A. nidulans amdS gene and recovered transformants able to use acetamide as a nitrogen source. Buxton et al., (Gene 37: 207, 1985) transformed A. niger argB.sup.- mutants with the A. nidulans argB.sup.+ clone.
There is a need in the art, however, for an efficient method of producing higher eucaryotic proteins in a system that can utilize established industrial fermentation technology. The present invention fulfills this need and further provides other related advantages.